Histochem Cell Biol (2008) 130:465–471
DOI 10.1007/s00418-008-0474-z
R EV IE W
Immunohistochemical assessment of protein phosphorylation
state: the dream and the reality
James W. Mandell
Accepted: 30 June 2008 / Published online: 22 July 2008
© Springer-Verlag 2008
Abstract The development of phosphorylation state-speciWc antibodies (PSSAs) in the 1980s, and their subsequent
proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks.
The extent to which this promise has been fulWlled is the
topic of this review. I review some applications of PSSAs
primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable
attention for their potential to reveal cell type-speciWc activation status, provide added prognostic information, aid in
the prediction of response to therapy, and most recently,
demonstrate the eYcacy of kinase-targeted chemotherapies.
However, despite some successes, many studies have failed
to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a
large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples
and their relationship to the actual phosphorylation states in
vivo. The next phase of translational research in applications
of PSSAs will entail the hard work of antibody validation,
gathering of detailed information about epitope-speciWc
lability, and implementation of methods for standardization.
Keywords PhosphospeciWc · Protein phosphorylation ·
Immunohistochemistry · Kinases
J. W. Mandell (&)
Department of Pathology,
University of Virginia School of Medicine,
P.O. Box 800904, Charlottesville, VA 22908, USA
e-mail: [email protected]
Introduction
Reversible protein phosphorylation was discovered over
50 years ago by Fischer and Krebs, during their studies on
the enzyme glycogen phosphorylase (Fischer and Krebs
1955). Phosphorylation and its reverse reaction, dephosphorylation, are carried out by over 500 kinases (Manning
et al. 2002) and a smaller number of phosphatases, respectively. An explosion of work on kinases, phosphatases and
their numerous protein substrates has generated an
immense body of biochemical knowledge; a current PUBMED search using the search term “phosphorylation” Wnds
over 150,000 articles. Until the development of phosphorylation state-speciWc antibodies (PSSAs), however, the study
of protein phosphorylation required radioactive phosphate
incorporation assays and was largely limited to biochemistry labs. PSSAs made possible the study of protein phosphorylation in situ, allowing cell biologists, histologists and
pathologists at least a static view of dynamic protein phosphorylation reactions in the spatially complex structures of
cells and tissues. In this review I focus on recent advances
in applications of PSSAs, focusing on studies of solid
tumors that include immunohistochemical approaches. I
will then address some technical and practical limitations in
the methodology, and suggest future directions to improve
the data arising from use of these reagents.
Historical aspects of the development of PSSAs were
discussed in a previous review (Mandell 2003). One key
reference omission in that article, however, was the Wrst
report of a PSSA, which recognized the phosphorylated
form of a substrate of cGMP-dependent protein kinase
(Nairn et al. 1982). This was one piece of a large body of
work by Paul Greengard’s group on protein phosphorylation in the nervous system, recognized with the awarding of
a Nobel prize in 2000 (De Camilli and Carew 2000).
123
466
The number of commercially available PSSAs has
steadily grown, as has the number of publications utilizing
these reagents in immunohistochemical applications
(Fig. 1). Despite this growth, there are still many unanswered questions about the robustness and reproducibility
of immunohistochemical assays of protein phosphorylation,
which will be discussed. The focus will be on immunohistochemical studies of human solid tumors over the past
5 years. Studies reviewed include novel morphological
observations on cellular signaling pathway activation, as
well as the potential for PSSAs to provide prognostic information, prediction of response to targeted therapies, and
post-treatment assessment of response to therapy. Due to
space limitations only a sampling of the large body of work
is discussed here.
The dream
In the realm of solid tumor assessment, the dream oVered
by PSSAs is the ability to provide robust multiparameter
measurements of intracellular signaling pathway status, in a
way that provides prognostic, predictive, and/or therapeutic
information to the clinician. An example of a multiparameter panel of PSSAs applied to a human glioblastoma is
shown in Fig. 2 (unpublished data). In this case, overexpression of total EGFR was present (compared to no signal
in adjacent nonneoplastic brain). In addition, strong tyrosine phosphorylation of the receptor was found at Tyr1173
but only focal weak phosphorylation at Tyr1068. Downstream kinases ERK and mTOR show strong cytoplasmic
Fig. 1 Continuous growth of publications with immunohistochemical
applications of PSSAs. A Google Scholar Advanced search (full text
search) using the Boolean search: {(“phosphospeciWc” OR “phosphospeciWc” OR “phosphorylation-speciWc” OR “phosphorylation statespeciWc”) AND “immunohistochemistry”} revealed a continuous
increase in the annual number of publications including these key
phrases in the full text content. The apparent slight decline in 2007 is
likely due to the restricted full text access of many journals in the Wrst
year after publication
123
Histochem Cell Biol (2008) 130:465–471
phosphorylation in a subset of tumor cells, whereas the
transcription factor CREB is strongly phosphorylated in the
majority of tumor cells. Phospho-Histone (H3) is an indicator of mitotic activity. Phosphorylation of the actin-associated proteins Ezrin/Radixin/Moesin, potential mediators of
tumor invasiveness, is strong in all cells. Finally, a generic
anti-phospho-tyrosine antibody is used to assess the general
preservation of tyrosine phosphorylation in this tumor sample. In the dream, we can provide quantitative measures of
protein phosphorylation, both in terms of percent of protein
phosphorylated, as well as in terms of percent cells positive
(above a stated threshold). Additionally, the analysis will
include information about subcellular localization of the
phosphoepitopes under study. All this information is then
entered into an algorithm that classiWes the tumor among
others of its general type, providing the oncologist with
robust and clinically useful information about the activation
proWle of signaling networks in this patient’s tumor. But
now we must awaken.
The reality
PSSAs as prognostic markers
A central question in the Weld is whether knowledge about
the phosphorylation (activation) state of a protein, in addition to its general level of expression, provides nonredundant information. Several studies on human solid tumors,
most focusing on EGFR family members, have begun to
address this question. Because immunohistochemistry for
total EGFR failed to demonstrate prognostic importance in
non-small cell lung carcinoma (NSCLC), it was hypothesized that information about the activation (phosphorylation) of EGFR could reveal prognostic signiWcance. To
address this, immunohistochemistry for total and phosphoEGFR as well as phospho-STAT3 was performed on archival NSCLC cases. Of the EGFR-expressing tumors,
approximately 1/3 showed pEGFR immunoreactivity, but
the presence of pEGFR did not provide statistically signiWcant prognostic information (Cortas et al. 2007).
In a similar study on locally advanced breast cancer,
total EGFR expression correlated with negative hormone
receptor status, and was associated with signiWcantly worse
relapse-free survival. However, the authors found no association of phospho-EGFR with outcome (Nieto et al. 2007).
A large body of evidence indicates that mutated BRAF
and NRAS contribute to the genesis and growth of melanomas,
presumably by activation of the extracellular signalregulated kinase (ERK) pathway. The presence of phosphorylated ERK1/2 was analyzed in 170 melanomas with
established NRAS/BRAF mutational status. The authors
found notable heterogeneity of phospho-ERK staining with
Histochem Cell Biol (2008) 130:465–471
467
Fig. 2 An example of a panel of phosphorylation state-speciWc antibodies applied to a human glioblastoma multiforme. a Overexpression
of total EGFR is apparent [(compared to little or no signal in adjacent
nonneoplastic brain, (not shown)]. In addition, strong tyrosine phosphorylation of the receptor was found at Tyr1173 (b) but only focal
weak phosphorylation at Tyr1068 (c). Downstream intracellular kinases ERK (d) and mTOR (e) show strong cytoplasmic and phosphoryla-
tion in a subset of tumor cells, whereas the transcription factor CREB
(f) is strongly phosphorylated in the nuclei of most tumor cells. Phospho-Histone H3 (g) is a marker of all cells in mitosis. Phosphorylation
of the actin-associated proteins Ezrin/Radixin/Moesin, potential mediators of tumor invasiveness, is strong in all cells (h). Finally, a generic
anti-phospho-tyrosine antibody reveals the general preservation of
tyrosine phosphorylation in this tumor section
only a minority of tumor cells positive in most cases. Moreover, the percentage of phospho-ERK-positive tumor cells
did not correlate with prognosis (Houben et al. 2008).
A frequently overlooked phenomenon is staining of nontumor stromal cells and the vasculature with PSSAs. A
large body of evidence points at the tumor microenvironment as critical to oncogenesis (Roskelley and Bissell
2002). In addition, vascular-targeted therapies are now in
common use, making PSSA assessment of stromal cell and
vascular cell signaling a possibly ripe area of investigation.
Mouse xenograft studies were performed with tumors that
produced TGF-alpha, stimulating endothelial cells to
express and activate EGFR (Kuwai et al. 2008). EGFR
blockade in these tumors lead to endothelial cell apoptosis
and subsequent tumor necrosis. Importantly, tumor xenografts in which TGF-alpha was knocked down with shRNA
did not stimulate endothelial EGFR activation, and these
tumors were resistant to therapy. Based on this and other
similar studies, the assessment of human tumor endothelial
cell EGFR, PDGFR or VEGFR activation status could provide clinically valuable information, but to date has not
been speciWcally analyzed.
Because the activation of mitogen-activated protein
kinase (MAPK) and members of the Akt pathway can promote glioblastoma cell proliferation, survival, and resistance to radiation, assessment of signaling pathway
activation was undertaken in a series of these tumors. Elevated p-MAPK was most strongly associated with poor
response to radiotherapy. Elevated p-mTOR, p-p70S6K,
and p-MAPK were associated with shorter survival. However, only p-MAPK proved to be an independent prognostic
factor after factoring for other clinical variables (Pelloski
et al. 2006).
Another study from the same group found that among
tumors lacking EGFRvIII expression (a truncated and
mutationally activated form of the receptor), the status of
phospho-AKT and phospho-ERK was prognostically signiWcant. However, when EGFRvIII was expressed these
markers did not provide additional prognostic power
(Pelloski et al. 2007). It seems likely that the EGFRvIIInegative tumors represent a more heterogeneous group
from the standpoint of molecular pathogenesis and thus
beneWt more from subclassiWcation using phospho-ERK
and phospho-AKT assessment.
123
468
Akt and ERK phosphorylation was investigated by
immunohistochemistry in patients with with lymph nodenegative breast cancer. P-Akt, but not p-ERK correlated
with HER-2/neu overexpression and was related to reduced
tumor apoptosis. No association was found between pAkt
or pERK with cell proliferation assessed by Ki67 and
mitotic count. P-Akt status proved to be a statistically signiWcant independent prognostic factor (Schmitz et al.
2004). Similarly, a study on head and neck squamous cell
carcinomas (HNSCC) found that Akt phosphorylation (Ser
473) correlates with poor prognosis (Massarelli et al. 2005).
Numerous studies have documented the overexpression
of the p53 tumor supressor protein in human tumors. P53
function is regulated not only by protein levels, but also by
phosphorylation state, which determines stabilization and
protection against mdm-2. With this in mind, a study investigated phosphorylation at Ser15 and Ser392 in squamous
carcinoma and precursor lesions. Although not tested for
prognostic value, the Wndings revealed diVerential staining
patterns with the two PSSAs, suggesting that high levels of
Ser392 phosphorylation is an early change in the pathogenesis of SCC (Matsumoto et al. 2004). However, a study on
gastroesophageal adenocarcinomas showed no correlation
of p53 phosphorylation (Ser15) with p53 mutation, and no
correlation with survival. In this setting, analysis of p53
phosphorylation did not appear to have obvious clinical
utility. (Puhringer-Oppermann et al. 2006).
A study on prostate cancers examined the phosphorylation Akt and relevant downstream substrates glycogen synthase kinase 3, mTOR, and the forkhead transcription
factor-like 1 (FKHRL1). Although most tumors showed
elevated phosphorylation of PKB/Akt in the malignant tissue compared with the surrounding benign tissue, only
increased phosphorylated-FKHRL1 levels correlated with
clinical progression (Jendrossek et al. 2008). Immunohistochemical analysis of renal cell carcinomas pAkt and pS6
was found to be independent prognostic factors (Pantuck
et al. 2007).
Because activation of Akt was documented in hormonerefractory prostate cancer (HRPC) and can result in phosphorylation of the androgen receptor (AR), IHC analysis of
the phosphorylation status of Akt and AR was tested in a
series of prostate cancer tumors. In the hormone-refractory
cases, phosphorylated AR (pAR) was associated with
worse prognosis (McCall et al. 2008).
Endocrine therapy is an important treatment option for
women with estrogen receptor (ER)-positive breast cancer.
Information about the activation state of ER could in theory
provide more clinically useful information than mere
protein expression. A study of ERalpha serine (Ser) 118
and ERalpha Ser167 revealed that low phosphorylation
of ERalpha Ser118 and high phosphorylation of
ERalpha Ser167 were associated with signiWcantly improved
123
Histochem Cell Biol (2008) 130:465–471
disease-free and overall survival. (Yamashita et al.
2008)This is a good example of another overlooked aspect,
namely careful assessment of dephosphorylation of speciWc
epitopes.
Overexpression or activation of the transcription factor
c-Jun has been implicated in the pathogenesis of several
types of cancer. Phospho-c-Jun staining was correlated
with signiWcantly shorter overall survival (Kuo et al.
2006).
PSSAs as predictive markers
A large number of studies have examined the role of immunohistochemical tests forErbB-2 (Her2/Neu) as predictive
markers for therapeutic responses of breast cancer to antiHer2 antibodies, culminating in an FDA-approved test
(Thor 2001). A logical hypothesis was that assessment of
the activation (tyrosine phosphorylation) of Her2 would
provide added predictive information. However, immunohistochemistry with the anti-phospho-ErbB-2 antibody had
no added beneWt to identify those patients most likely to
beneWt from increased doses of adjuvant chemotherapy
(DiGiovanna et al. 2008). However, a similar study but in a
diVerent subgroup of breast cancer patients did Wnd clinical
utility of PSSAs. The phosphorylation status of Her-2 and
EGFR was assessed in Her-2-overexpressing tumor samples from trastuzumab-treated metastatic breast cancers.
The presence of ptyr-1248 Her-2 and ptyr-845 or ptyr-1173
EGFR was a strong predictor response of both to anti-Her2
treatment. ptyr-845 EGFR and ptyr-1248 Her-2 were both
independent predictors of progression-free survival (Hudelist et al. 2006). The rapid growth of clinical trials of targeted chemotherapeutics will make the development and
validation of reliable PSSA IHC testing a high priority for
the next decade.
PSSAs as indicators of therapeutic eVect
An exciting area application for PSSAs is the assessment of
activity in post-treatment tumor samples. As a Wrst step
towards clinical application, animal tests were performed
using JNJ-10198409, an inhibitor of PDGFR in a nude
mouse xenograft model of human colon cancer. The
phosphorylation status of phospholipase Cgamma1
(PLCgamma1) was used as a marker of activation of the
PDGFR signaling cascade. By using a pair of antibodies in
serial sections, one for total (phosphorylated and unphosphorylated forms) and the other for the phosphorylated
form of PLCgamma1 (ph-PLCgamma1), an immunohistochemical ratio assay was achieved. Impressively, the
authors showed statistically signiWcant, dose-dependent
diVerences in the phospho/total PLCgamma1 ratio among
the four treatment groups (vehicle, 25, 50, and 100 mg/kg
Histochem Cell Biol (2008) 130:465–471
b.i.d.). This study should serve as a model for future work
in animal models as well as in the more complex and heterogeneous human samples (D’Andrea et al. 2005).
A neoadjuvant trial of rapamycin was recently completed in patients with recurrent glioblastoma, whose
tumors lacked expression of the tumor suppressor PTEN.
Several interesting observations were noted. The
magnitude of mTOR inhibition (measured by S6 protein
phosphorylation) varied substantially. Also, rapamycin
treatment led to Akt activation in seven patients, which was
associated with shorter time-to-progression (Cloughesy
et al. 2008).
Radiation therapy has profound eVects on cell signaling
pathways, underlying some aspects of its therapeutic activity. A study of ERK phosphorylation in colorectal cancer
revealed that most patients who were phospho-ERK positive before RT converted to ERK negativity after RT (Corn
et al. 2008). Although intriguing, further studies are needed
to determine if measurement of phospho-ERK can be predictive of response or an indicator of the eVect of to radiation therapy.
The use of surrogate cells, obtained noninvasively, to
assess chemotherapeutic activity would be a boon for
oncologists and much preferred by patients. Buccal mucosa
cells were tested as a model in which to assess geWtinib
activity in patients with advanced non-small cell lung cancer (Loprevite et al. 2007). Pretreatment and post-treatment
cells were tested for expression of p-EGFR, p-ERK and
p-AKT. Baseline p-AKT expression, but not that of EGFR,
p-EGFR, and p-ERK showed a potentially predictive role,
although the study did not reach statistical signiWcance.
Further work on buccal cells and other possible surrogate
cells will be a fruitful area.
New technologies
Quantitative image analysis
Like any immunohistochemical assay, a frequent bottleneck is the unbiased scoring of staining intensity, percent
positive cells, and subcellular localizations. Application of
an immunoXuorescence-based quantitative image analysis
system (AQUA) to the study of AKT phosphorylation
(Ser473) revealed the marker as a strong negative prognostic factor in oropharyngeal squamous cancer(Yu et al.
2007). Future applications of this and other image analysis
methods using ratio imaging of phospho- to total protein
immunoreactivity, as achieved with cultured cells (Mandell
and Banker 1996) could provide a readout of relative (%)
phosphorylation, more appropriate for comparisons of multiple samples in which the total amount of the phosphoprotein could vary.
469
FRET
Fluorescence resonance energy transfer (FRET) involves
the transfer of energy from an excited donor molecule to a
nearby (<7 nm) spectrally overlapping acceptor. Although
most widely applied to the study of interacting engineered
autoXuorescent proteins, FRET can be used to measure the
proximity of two appropriately labeled antibodies. Proofof-principle of this method was obtained in cancer cell lines
using the acceptor photobleaching Xuorescence resonance
energy transfer imaging approach to measure the phosphorylation state of EGFR (Keese et al. 2005). In a study on
head and neck cancers, a generic anti-phospotyrosine antibody and an anti-EGFR antibody were successfully used to
measure EGFR phosphorylation (Kong et al. 2006). FRET
eYciency correlated with worsening disease-free survival
but not with overall survival. FRET-based methods are
much more technically challenging than traditional immunohistochemistry but have some potential advantages. One
is the measurement of phosphorylation for proteins for
which no good PSSAs are available, by means of a pair of a
general (protein-speciWc) antibody and a generic anti-phosphotyrosine, phosphothreonine, or phosphoserine antibody.
The other is the inherent quantitative nature of the measurements. Finally, the method can potentially be used to measure the binding interactions of any pair of molecules in
tissue Wxed tissue samples.
The hard work ahead: antibody validation
and phosphoepitope characterization
An obvious need for PSSAs, as well as for the entire Weld
of immunohistochemistry, is improved antibody validation.
A good example of the multiple approaches necessary to
truly validate antibodies for quantitative immunohistochemistry was recently published for the case of C-Met
(Pozner-Moulis et al. 2007). Much more work will be
needed to bring the validation of a large set of PSSAs. Ideally, information gleaned from these types of studies can be
made available to the research community via public websites, such as the Abminer site hosted by the National Cancer Institute (http://discover.nci.nih.gov/abminer/).
Because reversible protein phosphorylation is inherently
labile, the use of PSSAs requires an additional set of assurances beyond those for general IHC antibodies. For example, surgically excised tissues are often kept at ambient
temperatures for long periods in the operating room or in
surgical pathology labs during the busy workday. A largely
unaddressed issue is the relative stability/lability of various
phosphoepitopes ex vivo. We initiated a pilot investigation
by growing xenografts in nude mice, excising the tumors,
and either Wxing immediately, or delaying Wxation for four
hours (Fig. 3). We were encouraged that some markers,
123
470
Histochem Cell Biol (2008) 130:465–471
Fig. 3 Ex Vivo phosphoepitope
lability: preliminary comparison
of phospho-NF-kappaB
(Ser276) and phospho-CREB
(Ser133). To model the potential
post-surgical loss of phosphoepitopes, tumor xenografts were
grown in nude mice, harvested,
and Wxed either immediately
(a, c), or 4 h later (b, d)
such as phospho-CREB (Ser133) showed no obvious loss
of phosphoepitope content at 4 h. However, others, such as
phospho-NFkappaB (Ser276) did show apparent loss of
immunoreacitivity especially in the interior portions of the
tumor. Much more work is needed to quantify phosphoepitope lability using both quantitative western blotting and
parallel IHC. If sentinel antibodies sensitive to the general
state of cellular phosphorylation levels can be identiWed, for
example, total phosphotyrosine, phosphothreonine, or
phosphoserine content, this could provide assurance of reasonable preservation of in vivo phosphorylation states.
Conclusions
The past 5 years has seen an explosion in the number of
immunohistochemical applications of PSSAs, especially in
the assessment of human solid tumors. Although it seems
logical that knowledge about protein phosphorylation state
should provide information above and beyond total protein
expression levels, a signiWcant number of studies have
shown no added beneWt of assessing phosphorylation compared to general antibody IHC. However, enough studies
do seem to indicate added prognostic, predictive, and
therapeutic monitoring information to warrant further
eVorts. Without better antibody validations and careful
characterization of individual phosphoepitopes in controlled model systems, it will not be possible to properly
123
interpret immunohistochemical results with these potentially powerful reagents.
Acknowledgments I thank George Glass, Peter Cummings, and
Tamara Stoops for providing pilot data. I thank Jason Papin, Erwin
Gianchandani (Dept. of Biomedical Engineering, U.Va.), David Brautigan and Michael Weber (Dept. of Microbiology) for helpful and provocative discussions.
References
Cloughesy TF, Yoshimoto K, Nghiemphu P, Brown K, Dang J, Zhu S,
Hsueh T, Chen Y, Wang W, Youngkin D, Liau L, Martin N,
Becker D, Bergsneider M, Lai A, Green R, Oglesby T, Koleto M,
Trent J, Horvath S, Mischel PS, MellinghoV IK, Sawyers CL
(2008) Antitumor activity of rapamycin in a phase I trial for patients with recurrent PTEN-deWcient glioblastoma. PLoS Med
5:e8
Corn BW, Kovner F, Bek S, Wexler I, Lifschits B, Seger R (2008)
ERK signaling in colorectal cancer: a preliminary report on the
expression of phosphorylated ERK and the eVects of radiation
therapy. Am J Clin Oncol 31:255–258
Cortas T, Eisenberg R, Fu P, Kern J, Patrick L, Dowlati A (2007) Activation state EGFR and STAT-3 as prognostic markers in resected
non-small cell lung cancer. Lung Cancer 55:349–355
D’Andrea MR, Mei JM, Tuman RW, Galemmo RA, Johnson DL
(2005) Validation of in vivo pharmacodynamic activity of a novel
PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis. Mol Cancer Ther
4:1198–1204
De Camilli P, Carew TJ (2000) Nobel celebrates the neurosciences.
Modulatory signaling in the brain. Cell 103:829–833
Histochem Cell Biol (2008) 130:465–471
DiGiovanna MP, Stern DF, Edgerton S, Broadwater G, Dressler LG,
Budman DR, Henderson IC, Norton L, Liu ET, Muss HB, Berry
DA, Hayes DF, Thor AD (2008) InXuence of activation state of
ErbB-2 (HER-2) on response to adjuvant cyclophosphamide,
doxorubicin, and Xuorouracil for stage II, node-positive breast
cancer: study 8541 from the Cancer and Leukemia Group B.
J Clin Oncol 26:2364–2372
Fischer EH, Krebs EG (1955) Conversion of phosphorylase b to phosphorylase a in muscle extracts. J Biol Chem 216:121–132
Houben R, Vetter-Kauczok CS, Ortmann S, Rapp UR, Broecker EB,
Becker JC (2008) Phospho-ERK staining is a poor indicator of
the mutational status of BRAF and NRAS in human melanoma.
J Invest Dermatol
Hudelist G, Kostler WJ, Czerwenka K, Kubista E, Attems J, Muller R,
Gschwantler-Kaulich D, Manavi M, Huber I, Hoschutzky H,
Zielinski CC, Singer CF (2006) Her-2/neu and EGFR tyrosine
kinase activation predict the eYcacy of trastuzumab-based
therapy in patients with metastatic breast cancer. Int J Cancer
118:1126–1134
Jendrossek V, Henkel M, Hennenlotter J, Vogel U, Ganswindt U,
Muller I, Handrick R, Anastasiadis AG, Kuczyk M, Stenzl A, Belka C (2008) Analysis of complex protein kinase B signalling pathways in human prostate cancer samples. BJU Int
Keese M, Magdeburg RJ, Herzog T, Hasenberg T, OVterdinger M,
Pepperkok R, Sturm JW, Bastiaens PI (2005) Imaging epidermal
growth factor receptor phosphorylation in human colorectal cancer cells and human tissues. J Biol Chem 280:27826–27831
Kong A, Leboucher P, Leek R, Calleja V, Winter S, Harris A, Parker
PJ, Larijani B (2006) Prognostic value of an activation state marker for epidermal growth factor receptor in tissue microarrays of
head and neck cancer. Cancer Res 66:2834–2843
Kuo RC, Lin CY, Kuo MY (2006) Prognostic role of c-Jun activation
in patients with areca quid chewing-related oral squamous cell
carcinomas in Taiwan. J Formos Med Assoc 105:229–234
Kuwai T, Nakamura T, Sasaki T, Kim SJ, Fan D, Villares GJ, Zigler
M, Wang H, Bar-Eli M, Kerbel RS, Fidler IJ (2008) Phosphorylated epidermal growth factor receptor on tumor-associated endothelial cells is a primary target for therapy with tyrosine kinase
inhibitors. Neoplasia 10:489–500
Loprevite M, Tiseo M, Chiaramondia M, Capelletti M, Bozzetti C,
Bortesi B, Naldi N, Nizzoli R, Dadati P, Kunkl A, Zennaro D,
Lagrasta C, Campanini N, Spiritelli E, Camisa R, Grossi F, Rindi
G, Franciosi V, Ardizzoni A (2007) Buccal mucosa cells as in
vivo model to evaluate geWtinib activity in patients with advanced
non small cell lung cancer. Clin Cancer Res 13:6518–6526
Mandell JW (2003) Phosphorylation state-speciWc antibodies: applications in investigative and diagnostic pathology. Am J Pathol
163:1687–1698
Mandell JW, Banker GA (1996) A spatial gradient of tau protein phosphorylation in nascent axons. J Neurosci 16:5727–5740
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002)
The protein kinase complement of the human genome. Science
298:1912–1934
Massarelli E, Liu DD, Lee JJ, El-Naggar AK, Lo Muzio L, Staibano S,
De Placido S, Myers JN, Papadimitrakopoulou VA (2005) Akt
activation correlates with adverse outcome in tongue cancer.
Cancer 104:2430–2436
471
Matsumoto M, Furihata M, Kurabayashi A, Ohtsuki Y (2004) Phosphorylation state of tumor-suppressor gene p53 product overexpressed in skin tumors. Oncol Rep 12:1039–1043
McCall P, Gemmell LK, Mukherjee R, Bartlett JM, Edwards J (2008)
Phosphorylation of the androgen receptor is associated with
reduced survival in hormone-refractory prostate cancer patients.
Br J Cancer 98:1094–1101
Nairn AC, Detre JA, Casnellie JE, Greengard P (1982) Serum antibodies that distinguish between the phospho- and dephospho-forms
of a phosphoprotein. Nature 299:734–736
Nieto Y, Nawaz F, Jones RB, Shpall EJ, Nawaz S (2007) Prognostic
signiWcance of overexpression and phosphorylation of epidermal
growth factor receptor (EGFR) and the presence of truncated EGFRvIII in locoregionally advanced breast cancer. J Clin Oncol
25:4405–4413
Pantuck AJ, Seligson DB, Klatte T, Yu H, Leppert JT, Moore L, O’Toole T, Gibbons J, Belldegrun AS, Figlin RA (2007) Prognostic
relevance of the mTOR pathway in renal cell carcinoma: implications for molecular patient selection for targeted therapy. Cancer
109:2257–2267
Pelloski CE, Lin E, Zhang L, Yung WK, Colman H, Liu JL, Woo SY,
Heimberger AB, Suki D, Prados M, Chang S, Barker FG 3rd, Fuller GN, Aldape KD (2006) Prognostic associations of activated
mitogen-activated protein kinase and Akt pathways in glioblastoma. Clin Cancer Res 12:3935–3941
Pelloski CE, Ballman KV, Furth AF, Zhang L, Lin E, Sulman EP, Bhat
K, McDonald JM, Yung WK, Colman H, Woo SY, Heimberger
AB, Suki D, Prados MD, Chang SM, Barker FG 2nd, Buckner JC,
James CD, Aldape K (2007) Epidermal growth factor receptor
variant III status deWnes clinically distinct subtypes of glioblastoma. J Clin Oncol 25:2288–2294
Pozner-Moulis S, Cregger M, Camp RL, Rimm DL (2007) Antibody
validation by quantitative analysis of protein expression using
expression of Met in breast cancer as a model. Lab Invest
87:251–260
Puhringer-Oppermann F, Stahl M, Keller G, Sarbia M (2006) Lack of
prognostic impact of p53 gene mutation and p53 phosphorylation
at serine 15 in multimodally treated adenocarcinomas of the gastroesophageal junction. J Cancer Res Clin Oncol 132:433–438
Roskelley CD, Bissell MJ (2002) The dominance of the microenvironment in breast and ovarian cancer. Semin Cancer Biol 12:97–104
Schmitz KJ, Otterbach F, Callies R, Levkau B, Holscher M, HoVmann
O, Grabellus F, Kimmig R, Schmid KW, Baba HA (2004) Prognostic relevance of activated Akt kinase in node-negative breast
cancer: a clinicopathological study of 99 cases. Mod Pathol
17:15–21
Thor A (2001) HER2—a discussion of testing approaches in the USA.
Ann Oncol 12(Suppl. 1):S101–S107
Yamashita H, Nishio M, Toyama T, Sugiura H, Kondo N, Kobayashi
S, Fujii Y, Iwase H (2008) Low phosphorylation of ER{alpha}
serine 118 and high phosphorylation of ER{alpha} serine 167
improve survival in ER-positive breast cancer. Endocr Relat Cancer
Yu Z, Weinberger PM, Sasaki C, Egleston BL, Speier WFT, HaVty B,
Kowalski D, Camp R, Rimm D, Vairaktaris E, Burtness B, Psyrri
A (2007) Phosphorylation of Akt (Ser473) predicts poor clinical
outcome in oropharyngeal squamous cell cancer. Cancer Epidemiol Biomarkers Prev 16:553–558
123